Various recombinant human proteins are produced using mammalian cell culture systems that over-express and secrete these biologics into the medium which are then purified by chromatographic processes. Successfully produced recombinant proteins include secretory proteins (e.g., blood clotting factors, immunoglobulins, erythropoietin and other hormones, elastase inhibitors, etc.) and lysosomal enzymes (e.g., β-glucocerebrosidase, α-galactosidase A, acid α-glucosidase, etc.). Several critical factors impact the efficiency and yield in such a manufacturing process: the level of expression for cell line; whether the secreted recombinant protein maintains its biological activity in the cell culture medium prior to purification; and the protein purification scheme for recovery of the biologic.
The above examples are proteins which all share a common biosynthetic pathway at the endoplasmic reticulum (ER) (Blobel et al., 1979). These proteins (which include all membrane proteins, secretory proteins, peroxisomal and lysosomal proteins) also share a common export pathway out of the ER which traffics the newly synthesized proteins to the Golgi apparatus for additional post-translational modifications to sort different classes of proteins so that they reach their intended cellular and extracellular destinations (Kornfeld, 1987). In order for these proteins to reach their final destinations, they must first fold into stable structures that sufficiently pass the ER quality control (QC) system prior to exiting this compartment (Ellgaard & Helenius, 2003). Mutant proteins often do not fold stably and are recognized by the ER QC system and retained (Ellgaard & Helenius, 2003). If these mutant proteins fail to reach a stable conformation after multiple attempts, they are ultimately eliminated by the ER-associated degradation (ERAD) systems. Aberrant ER retention of less stable mutant proteins and excessive ERAD have been shown to be the primary cause of numerous diseases including cystic fibrosis, type 2 diabetes, and various lysosomal storage diseases (Schmitz et al., 2005; Fan et al., 1999; Tropak et al., 2004).
Premature degradation of normal proteins is also observed such that an appreciable fraction of wild-type proteins fails to reach stable conformations within the allotted timeframe and ultimately eliminated by ERAD. The most cited example is the elimination of 50-70% of the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) chloride ion channel. It is believed that large, complex proteins (e.g., CFTR, receptors, clotting factors, etc.) tend to fold less efficiently than smaller, simpler counterparts and are therefore prone to premature degradation. Moreover, Randall Kaufman and colleagues described the accumulation of recombinant human Factor VIII and unusual swelling of the ER, acute activation of certain kinases and various cellular pathways during the production of this biologic. These profound cellular effects are now known as ER stress associated with the expression of complex proteins and other problematic proteins (e.g., Factor VIII and Z-form alpha-1 antitrypsin). It is also believed that protein accumulation, excessive degradation and ER stress adversely affects recombinant protein production and lead to low protein yields. Thus there exists a need to improve the manufacturing process of recombinant proteins.